1. Field of the Invention
This invention relates to an apparatus for use in performing tests to determine the blood type of a donor or patient according to the ABO classification system.
In our knowledge of blood physiology we know that the differences between the red blood cells of individuals lie in the chemical structures on the cell surfaces. These structures are called blood group antigens and are the result of inherited genes. We are also aware that the body produces antibodies which attack and destroy substances foreign to the host body. This is the underlying factor in the immunological reactions carried on in the human body. In fact the production of an antibody is stimulated by the introduction of the foreign substance. Foreign substances which stimulate the production of antibodies are termed antigens, and blood group antigens lead to the production of antibodies that will destroy the antigens and the red cells of which they are a part.
Inasmuch, as noted above, as the red blood cells of different individuals have different antigens, it is clear that in an emergent situation involving life or death from a trauma, on-site determination of blood type may be necessary for proper transfusion. If, in transfusing blood to a patient, the transfused donor's red blood cells had antigens different than those of the patient, the introduction of the donor's blood into the patient's blood stream would immediately stimulate the production of antibodies that would destroy the transfused blood and thereby vitiate the effects which it was desired to give the patient by the transfusion. It needs no emphasis to say that the consequences could be dire. On the other hand, if the red blood cells of the donor and the patient each had the same antigens, blood could be transfused from one to the other without any untoward incidents.
The foregoing is very well known, and it has led to a blood classification system based on the presence of the known blood group antigens. The principal blood group antigens are designated A and B, with some red cells having A antigens, some having B antigens, some having both A and B antigens, and some having neither antigen. These blood groups are designated, respectively, Type A, Type B, Type AB, and Type O. There are other genetic factors which have to be taken into account, but in general the foregoing blood group types offer a satisfactory preliminary categorization to help insure safe blood transfusions.
There is one other antigen that is significant and the presence or absence of which must be determined, and that is the Rh antigen. As are the other antigens referred to, it is an important factor in blood transfusions, but is especially important in pregnancy where an Rh negative mother who developed Rh antibodies during a previous pregnancy may transfer those antibodies to a fetus having Rh positive blood with possibly fatal results to the fetus or to the baby shortly after birth.
Blood cell compatibility is determined by the non-occurrence of an immunological reaction between antibodies contained in the blood serum of a patient and antigens present on blood cells from a donor. For example, if the red cells of patient are type A (i.e., have "A" antigens on the red cells), the serum of such a patient's blood will have anti-B antibodies, i.e., antibodies which will react with "B" blood, an immunological reaction will occur between the anti-B antibodies of the patient's serum and the B-antigens of the red blood cells of the donor. Such an incompatibility can result in intra-vascular hemolysis.
Test for blood cell typing and compatibility are generally of two types: (i) a test to determine whether a specific antibody added to the cells will cause their agglutination, and (ii) a test to determine whether a specific antibody added to the tested cells together with serum complement, will cause cell lysis.
The first of these two basic tests, agglutination, refers to a clumping of blood cells containing, for example, type A antigens, to which anti-A antibodies are added in the absence of complement. The A-antigen and the anti-A antibody react specifically with each other by immunological reaction with the antibody forming bridges between adjacent cells. This leads to an interlocked mass of the blood cells joined to each other by the added antibodies. This mass may be easily visually observed.
The second of the two tests referred to above, cell lysis, relates to the disruption of cell membranes leading to death of the cells and release of their intracellular contents. Cell lysis is the result of a reaction which occurs between cell membrane bound antibody and a group of potentially destructive proteins in normal serum (called "complement").
Both methods described above are used for the typing and compatibility testing of the cellular blood elements, erythrocytes, granulocytes, B and T lymphocytes, and platelets (thrombocytes). Both methods are intrinsically qualitative and each may be used separately for assay of antigen, antibody, and serum complement.
2. Description of the Prior Art
In blood cell typing and compatibility test procedures commonly used today, both agglutination tests and cell lysis tests are carried out in a liquid phase, that is, sera containing antibodies with or without complement to be tested are mixed with suspensions of the blood cells with respect to which blood typing or compatibility testing is to be evaluated. Normally, fixed volumes are employed.
Evaluation of agglutination test results requires the technician to distinguish agglutination of cells due to specific antigen-antibody molecular bridging from non-specific cell aggregation in which unrelated forces also cause some degree of clumping. The technician must also be able to distinguish free unagglutinated cells which may be present from clumped or agglutinated cells. This requires highly experienced personnel or precise particle sizing and counting with costly instruments. In addition, measurement of the degree of specific agglutination is either poorly semi-quantitative or is costly and complicated to perform.
While some instrumented tests for typing of red blood cells by agglutination have been developed, the equipment for these procedures is both costly and complicated to use. For example, one device which has been proposed for typing red blood cells by instrument is known as the "Auto Analyzer" of Berkman et al. described in Transfusion, Vol. 11, No. 6, pp. 317 et seq. (1971), and of Rosenfeld et al. described in Vox. Sang. 26:289-333, 1974. In the AutoAnalyzer, blood samples and antibody sera are combined under special circumstances in complex tubular coils designed to bring about agglutination. The sample from the reaction coils passes a "T" connection with the leg in a downward position so that agglutinates which are formed tend to be removed. Agglutination can be detected by measuring the decrease in optical density of the effluent from the "T" carrying the non-agglutinated fraction (Berkman et al. and Rosenfield et al.), or by trapping very strong agglutinates from the "T" on filter paper (Shield et al., Transfusion, Vol. 9, p. 348, 1969).
An alternative device is known as the "Groupmatic" and can cost several hundred thousand dollars (see Garretta et al., Vox Sang., Vol 27, p. 141, 1974). In the Groupmatic device, sera and blood cell suspensions are combined to produce agglutination. The presence of agglutination is detected by passing the suspension across two light beams, one of which passes through the center of the reaction cuvette while the other passes through the periphery. A difference in the transmission of the beams is taken as the measure of the strength of agglutination. Sophisticated circuitry is required, however, placing the instrument beyond use in the field by emergency personnel.
All liquid-phase hemagglutination tests, be they manual, "AutoAnalyzer", or "Groupmatic", suffer from a series of problems. Firstly, the manual tests lack the sensitivity of the instrumented tests so that clinically-significant red cell antigen-antibody reactions may be indiscernible by even the most experienced personnel. Secondly, unnecessary and unwanted protein in the reaction mixture may actually interfere with the development of hemagglutination, and cannot be removed readily without significant loss or elution of antibody. Thirdly, the red cells of about 2% of persons are unsuitable for sensitive evaluation by AutoAnalyzer (and presumably also by Groupmatic). Fourthly, red cells lose their sensitivity to specific hemagglutination on storage at 4.degree. C., even for 2-3 weeks. Fifthly, not even the sensitive methods of Berkman et al. and Rosenfield et al. will detect all clinically-significant red cell antigen-antibody reactions. Lastly, "AutoAnalyzer" and "Groupmatic" tests are inefficient, lack portability, require stores of reagents, and are extremely tedious and can only be performed manually in a laboratory by the most experienced personnel.
These problems affect emergency blood typing where even routine red cell typing remains a time-consuming, manual operation that demands more skillful and experienced personnel than are available.
Hartnett, et al. in U.S. Pat. No. 3,905,772 describes an apparatus for performing blood typing tests which utilizes a plurality of test tubes formed as an integral unit together with a member that is marked vendorcators for reagents to be mixed in the tubes in performing the necessary tests to determine the blood type groups of various specimens. The apparatus is not portable and is not adapted for field use. Further, the apparatus is not self-contained and reagent and serum must be added to the blood samples and the test tubes.
In U.S. Pat. No. 4,275,053 issued June 23, 1981, Rosenfield, et al. describes a blood cell typing and compatibility procedure based upon either agglutination or immune lepis. A solid matrix is utilized and brought into contact with a serum containing the necessary antibodies for testing. Similarly, Barr in U.S. Pat. No. 4,252,538 issued Feb. 24, 1981, describes an apparatus and method for antibodies screening, typing and compatibility testing of red blood cells wherein a dual cavity substrate having a sample compartment for selectively discharging the blood specimen under test is rotably mounted in a holder. In the test procedure, a mono layer of red blood cells is centrifugally developed on the cavity surfaces of the substrate and subsequently typed.
Bartos, et al. in U.S. Pat. No. 4,162,033 describe a ready-for-use rapid test package for serological tests or in typing as carried out with preserved reactants in solid form in quantity suitably adjusted to each other. This invention utilizes reagents in ampules and makes possible an extraordinarily large number of serological investigations to a wide circle of users. The Bartos rapid test package makes possible screening of a large number of patients without the need to access large diagnostic laboratory facilities. The reactants in the ampules are preserved in frozen or freeze dried form for example
Thus it is clear that the classification of a person's blood type must sometimes be done in an emergency yet the possibility of wrongful classification minimized, if not eliminated. This is especially true under out of the hospital situations involving disasters such as plane or train crashes where blood has to be transfused to a number of individuals sometimes after on the spot grouping.
In performing a blood grouping test, it is usual to test an individual's blood cells for the presence of antigens. Usually, a multiplicity of sample-containing test tubes must be used for the tests on an individual's blood. In a disaster area where numerous blood specimens must be typed simultaneously, the typing function involves many pipettings and agglutination observations that involve blood and reagents for each patient specimen. Such functions are not conductive to a field environment. Unless the utmost care is exercised at all times there is the danger that a test tube and contents associated with one individual will be interchanged with that of another or become contaminated. Even with the exercise of care, there is always the possibility of an inadvertent mix-up of test tubes, reagents, and contents being tested.
A major cost factor in making a determination of blood type according to the ABO classification system is the cost of a technican's time in mixing and distributing various reagents for individual blood grouping tests. Often times, errors result from the incorrect mixing of an inappropriate reagent with a blood sample.